A subject of the present invention is a new process for the preparation of Fexofenadine.
Fexofenadine is a medicament which has a significant antihistaminic activity and which is free from side effects. (Efficacy and safety of Fexofenadine hydrochloride for treatment of seasonal allergic rhinitis, Bernstein D. I. et al, Ann. Allergy-Asthma-Immunol. (1997) 79(5) 443-448).
This compound is the principal metabolite of Terfenadine, itself an antihistaminic agent (D. Mc Tavish, K L Goa and M. Ferill, xe2x80x9cterfenadine: an updated review of its pharmacological properties and therapeutic efficiency, Drug 39, 552-574 (1989)).
Fexofenadine is currently prepared by chemical route, in numerous stages with a yield of less than 10% (U.S. Pat. No. 5,578,610 and 5,581,011).
The Applicant therefore proposes to find another synthesis route for Fexofenadine. The choice is then concentrated on a bioconversion method using one of the following very specific two types of microorganisms: either the filamentous fungi of the Absidia Corymbifera genus and in particular Absidia Corymbifera LCP 63-1800, or a Streptomyces and in particular Streptomyces platensis NRRL 2364. The specificity of the bioconversion with these microorganisms was unexpected: among the numerous strains studied during a screening, these are the only ones which allow Fexofenadine to be obtained with a good yield and little or no by-products.
A subject of the invention is a process for the preparation of the compounds of formula (I): 
characterized in that a bioconversion of the compound or compounds of formula (II) is carried out: 
with either a microorganism culture of the Absidia corymbifera genus or a microorganism culture of the Streptomyes genus, at a pH comprised between 5.0 and 8.0, in order to obtain the expected compound or compounds of formula (I), which if appropriate are isolated, purified and/or salified, the compounds of formulae (I) or (II) being able to be in the two possible enantiomeric forms, isolated or in mixtures.
The asterisk indicates the position of the asymmetrical carbon.
A particular subject of the invention is a preparation process as described above in which the Absidia corymbifera is Absidia corymbifera LCP 63-1800 or in which the Streptomyces is Streptomyces platensis NRRL 2364.
Fexofenadine is a racemic mixture of the enantiomers of formula (I). A more particular subject of the process is therefore the process as described previously in which a bioconversion of Terfenadine, corresponding to a racemic mixture of the two enantiomers of formula (II), is carried out in order to obtain Fexofenadine, corresponding to a racemic mixture of the enantiomers of formula (I).
Absidia corymbifera and in particular Absidia corymbifera LCP 63-1800 are available from the Laboratoire de Cryptogamie du Museum d""Histoire Naturelle de Paris [The Cryptogam Laboratory at the Paris Natural History Museum].
Among the Streptomyces which can be used in the process, which is a subject of this Application, the following Streptomyces can be mentioned:
Streptomyces albus 
Streptomyces ambofaciens ATCC 15154
Streptomyces antibioticus ATCC 31771
Streptomyces aureofaciens ATCC 10762
Streptomyces djakartensis 
Streptomyces erythraeus 
Streptomyces felleus DSM 40130
Streptomyces fradiae W3554
Streptomyces griseus NRRL B150
Streptomyces JSP-2 (FH2126)
Streptomyces lividans JT46/pCS2
Streptomyces narbonensis FH 2102
Streptomyces olivaceus ATCC 3335
Streptomyces platensis ATCC 13865
Streptomyces platensis NRRL 2364
Streptomyces rimosus 2234
Streptomyces venezuelae NRRL B-2446.
It is known to a person skilled in the art that the extreme simplicity of bacterial cells, without a distinct nucleus, allows them to be classified as prokaryotes. This is the case for Streptomyces, filamentous bacteria, which are aerobes and gram-positive. On the other hand, the other microorganisms are called eukaryotes suchas, for example, the filamentous fungi and quite particularly Absidia corymbifera. Their cells are differentiated in particular from the prokaryotes by the presence of a nucleus and numerous cytoplasmic organelles.
The process described above, which is a subject of the present Application, offers numerous advantages. On the one hand, it avoids the chemical route which requires numerous synthesis stages accompanied by necessary isolation processes at each of these reaction stages. In the case of an industrial use, this route can prove to be expensive and polluting.
In the case of the process which is a subject of this Application, a single operation is necessary and the optional purification stage essentially only has the purpose of eliminating a by-product which can form during the bioconversion, namely triolphosphate which corresponds to the alcohol non yet oxidized to acid, which is esterified in the form of a phosphate (formula (IIIb)) described below.
On the other hand this process lends itself to industrial use. By operating with a concentration of starting product of 0.5 g/l, the yield exceeds 70% relative to the starting product.
Among the other advantages of the use of the microbiological route against the chemical route, the non polluting aspect of this technique can be emphasized, all the operations taking place in aqueous media.
The purification is carried out according to the methods known to a person skilled in the art. It can be purification by crystallization, by chromatography or by ion exchange resin.
The salification reactions can be carried out under the usual conditions. For example the operation can be carried out in the presence of ethanolic soda. A sodium salt can also be used such as sodium or potassium carbonate or acid carbonate. The salts obtained can be the salts of alkali or alkaline-earth metals or optionally substituted ammonium.
The implementation of the oxidation is carried out according to the methods which are currently used for the microbiological oxidation of organic molecules using cultures of filamentous fungi (Holland HL, Organic synthesis with oxidative enzymes. VCH publisher, Inc, New York 1992; Lacroix I, Biton J and Azerad R, Microbial biotransformation of a synthetic immunomodulating agent HR325, Bioorg. Med. Chem. (1997) 7, 1369-1380; Sebek O K, Fungal transformations as a useful method for the organic synthesis of organic compounds, Mycologia 75(2) 383-394, 1983; Azerad, R. Microbial models for drug metabolism, Advances in Biochemical Engineering and Biotechnology, Vol. 63, page 169, 1999.
Thus, firstly, the most favourable fermentation conditions are determined by analytical route, in particular by thin layer chromatography or HPLC, in prior tests, such as for example the choice of the nutrient medium, the solvent of the appropriate substrate, the concentration of substrate, the technical conditions such as temperature, aeration, pH, and the optimum periods for the culture, addition of the substrate and contact of the substrate with the microorganism.
In a first phase, the culture is carried out from an inoculum (spores or mycelium) of Absidia corymbifera LCP 63-1800 or a bacteria of the Streptomyces genus, in particular Streptomyces platensis NRRL 2364, in a liquid nutrient medium at an initial pH of 5 to 7 and at a temperature of 20 to 30xc2x0 C., preferably from 26 to 28xc2x0 C. Aeration is ensured by rotary agitation of the culture receptacles (150 to 250 rpm) or, in the fermentation vessel, by the introduction of air at a flow rate of approximately 1 l/min and per litre of broth culture medium.
After a time varying from 24 to 72 hours, preferably 60 to 65 hours, the terfenadine is added at a concentration of 0.1 to 1 g/litre, preferably 0.4 to 0.6 g/litre, in solution in an organic solvent which is miscible with water, such as ethanol, acetone, tetrahydrofuran, dimethylformamide or dimethylsulphoxide (5 to 20 ml/litre of culture), preferably ethanol (10 ml/litre). The incubation is continued under the same conditions as the culture. The pH is optionally readjusted and maintained at a value of 5.0 to 8.0. The conversion of the substrate is advantageously followed by HPLC analysis of the incubation supernatant or thin layer chromatography of samples extracted with an organic solvent. In general after 48 to 200 hours, sufficient quantities of Fexofenadine have been formed.
Another procedure consists of separating the biomass of the culture medium by filtration, washing it with water or a solution buffered to a neutral pH, then resuspending it in a suitable buffer, for example a 0.05 M phosphate buffer at pH 7, then adding the substrate and continuing the incubation as described previously.
The isolation and purification of the products of the process is carried out in a manner which is known per se. For example, the products of the process can be extracted with an organic solvent, preferably ethyl acetate, or by adsorption on a hydrophobic column, followed by elution with an organic solvent, evaporating the organic solvent, separating and purifying the products by column chromatography or by crystallization.
Therefore a more particular subject of the invention is the process as described previously in which the biotransformation conditions are as follows: concentration of terfenadine added from 0.5 g/l to 10 g/l and preferably from 0.5 g/l to 5 g/l, pH evolving between 5.0 and 8.0, temperature comprised between 26xc2x0 C. and 28xc2x0 C. and aeration by the introduction of an air flow of approximately 1 1/min. and per litre of culture broth medium.
One of the objectives of the invention is to adjust the pH during the incubation and optionally to maintain it at an optimized value, with a view to minimizing the formation of by-products.
Conversion of the Terfenadine (II) to Fexofenadine (I) by A. corymbifera or S. platensis involves several successive stages, with the intermediate formation of the triol (IIIc) and that of two undesired by-products, Terfenadine-phosphate (IIIa) and triolphosphate (IIIb) according to the diagram below: 
The initial oxidation of terfenadine (I) to triol (IIIc), produced by the microorganism in the incubation medium, is encouraged by a slightly acid or neutral pH (6 less than pH less than 7) (Table 2). The formation (apparently irreversible) of Terfenadine-phosphate (IIIa) remains very limited in all the conditions studied and less than 1-2%. On the other hand, the formation of triol-phosphate (IIIb) represents a significant part of the oxidation products accumulated in the medium, particularly when the incubation is carried out at a neutral or alkaline pH (pH 7.0-8.0) as shown in Table 3 (see also FIG. 1); this is the pH towards which the incubation medium naturally evolves. However, at a slightly more acid pH (optimum pHxcx9c6), a phosphatase of fungal origin, present in the incubation medium, catalyzes the rapid hydrolysis of the triol-phosphate (IIIb) (Table 4, FIG. 1), whilst the irreversible oxidation of the triol to fexofenadine is encouraged at a higher pH (pH greater than 7) (Table 5, FIG. 1).
As a result there are several possible strategies for transforming almost all of the Terfenadine to Fexofenadine (if one does not take into account the very small quantity of Terfenadine-phosphate formed):
firstly proceeding with incubation in the initial culture medium at a pH of approximately 6.5, without monitoring the pH, which leads (at the same time as the pH evolves towards 8.0-8.5) to a complete transformation of the terfenadine to a stable stationary mixture of triol-phosphate (IIIb) and fexofenadine (FIG. 2) (Examples 2 and 3), then lowering the pH to a value comprised between 3.5 and 4 in order to encourage the transformation of (IIIb) to (IIIc) and allowing it to slowly and spontaneously rise towards 6.0, until the complete disappearance of the triol-phosphate (IIIb), then readjusting and maintaining the pH in the region of 8.0, until transformation of the triol (IIIc) to fexofenadine (FIG. 3) (Example 4).
proceeding in the same manner with an incubation in the culture medium, initially at a pH of approximately 6.5, without monitoring the pH, (the pH naturally evolves towards 8.0-8.5) then lowering and maintaining it at a value comprised between 6.0 and 6.5, at which pH the triol-phosphate (IIIb) is quite rapidly hydrolyzed, as a result of the equilibrium of phosphatase-phosphorylase activities at this pH, whilst the triol (IIIc) is itself quite rapidly oxidized, until virtually complete transformation to fexofenadine (FIG. 4) (Example 5).
A more particular subject of the invention is therefore:
a process as defined above in which the initial pH at approximately 6.5,
naturally evolves to 8.0-8.5, which leads to a mixture of triol-posphate (IIIb) and a compound of formula (I),
is then lowered to a value comprised between 3.5 and 4 in order to encourage the transformation of the intermediate compound of formula (IIIB) to the intermediate compound of formula (IIbc),
then naturally evolves to approximately 6.0 until the complete disappearance of compound (IIIb),
and finally is readjusted and maintained at approximately 8.0 until transformation of compound (IIIc) to Fexofenadine.
a process as defined above in which the initial pH is approximately 6.5
naturally evolves to 8.0-8.5,
is then lowered and maintained at a value comprised between 6.3 and 6.8.
a process as defined above in which during the purification stage, extraction of the product of formula (I) is carried out in the ethyl acetate.
Finally, a subject of the invention is also, as intermediate compound, the compound of formula (IIIc), as defined above.
The following examples illustrate the invention without however limiting it.
Preparation of the Different Culture Media
Medium A: Corn steep liqueur, 10 ml; NaNO3, 2 g; MgSO4 7H2O, 0.5 g; FeSO4.7H2O, 0.02 g; KCl, 0.5 g for 900 ml of distilled water. Phosphate buffer (K2HPO4, 2 g; KH2PO4, 1 g in 40 ml of distilled water) and glucose, 30 g in 60 ml of distilled water, added at the moment of seeding.
Medium B: Soya peptone, 5 g; yeast extract, 5 g; MgSO47H2O, 0.5 g; NaNO3, 2 g; KCl, 0.5 g; FeSO4.7H2O, 0.02 g for 900 ml of distilled water. Phosphate buffer (K2HPO4, 2 g; KH2PO4, 1 g in 40 ml of distilled water) and glucose, 30 g in 60 ml of distilled water, added at the moment of seeding.
Medium C: Soya peptone, 5 g; yeast extract, 5 g; NaCl, 5 g; K2HPO4, 5 g for 900 ml of distilled water. Glucose, 100 ml of a solution at 200 g/L added at the moment of seeding.
Medium D: Corn steep liquor, 10 ml; soya peptone, 5 g; NaNO3, 2 g; MgSO4.7H2O, 0.5 g; FeSO4.7H2O, 0.02 g; KCl, 0.5 g for 900 ml of distilled water. Phosphate buffer (K2HPO4, 2 g; KH2PO4, 1 g in 40 ml of distilled water) and glucose, 30 g in 60 ml of distilled water, added at the moment of seeding.
Medium E: Medium C adjusted to pH 7 with concentrated HCl, before sterilization.